Multiple respiratory viruses, including influenza A virus (IAV), can be transmitted via expiratory
aerosol particles, and
aerosol pH was recently identified as a major factor influencing airborne virus infectivity. Indoors, small exhaled
aerosols undergo rapid acidification to pH ~4. IAV is known to be sensitive to mildly acidic conditions encountered within host endosomes; however, it is unknown whether the same mechanisms could mediate viral inactivation within the more acidic
aerosol micro-environment. Here, we identified that transient exposure to pH 4 caused IAV inactivation by a two-stage process, with an initial sharp decline in infectious titers mainly attributed to premature attainment of the post-fusion conformation of
viral protein haemagglutinin (HA).
Protein changes were observed by
hydrogen-
deuterium exchange coupled to mass spectrometry (HDX-MS) as early as 10 s post-exposure to acidic conditions. Our HDX-MS data are in agreement with other more labor-intensive structural analysis techniques, such as X-ray crystallography, highlighting the ease and usefulness of whole-virus HDX-MS for multiplexed
protein analyses, even within enveloped viruses such as IAV. Additionally, virion integrity was partially but irreversibly affected by acidic conditions, with a progressive unfolding of the internal matrix
protein 1 (M1) that aligned with a more gradual decline in viral infectivity with time. In contrast, no
acid-mediated changes to the genome or
lipid envelope were detected. Improved understanding of respiratory virus fate within exhaled
aerosols constitutes a global public health priority, and information gained here could aid the development of novel strategies to control the airborne persistence of seasonal and/or pandemic
influenza in the future. IMPORTANCE It is well established that
COVID-19,
influenza, and many other
respiratory diseases can be transmitted by the inhalation of aerosolized viruses. Many studies have shown that the survival time of these airborne viruses is limited, but it remains an open question as to what drives their infectivity loss. Here, we address this question for influenza A virus by investigating structural
protein changes incurred by the virus under conditions relevant to respiratory aerosol particles. From prior work, we know that expelled
aerosols can become highly acidic due to equilibration with indoor room air, and our results indicate that two
viral proteins are affected by these acidic conditions at multiple sites, leading to virus inactivation. Our findings suggest that the development of air treatments to quicken the speed of
aerosol acidification would be a major strategy to control infectious bioburdens in the air.